Development of a broadly effective vaccine against diarrhea caused by enterotoxigenic Escherichia coli (ETEC) Project Summary: In this collaborative project between Johns Hopkins and South Dakota State University, we propose developing a broadly effective vaccine to protect against the most common bacterial cause of acute watery diarrhea in the world, enterotoxigenic Escherichia coli (ETEC). In developing countries ETEC cause 280 - 400 million diarrhea cases in children < 5 years, 100 million cases in children over 5 years, and 400 million cases in adults, including travelers and deployed military personnel. Among children, ETEC kill about 300,000 of the 1.3 million diarrhea deaths annually. These infections in children lead to stunting and poor cognitive development placing a burden on society as well as the child. ETEC actually represent a group of Escherichia coli of different serotypes that produce one or both enterotoxins known as heat- labile and heat-stable enterotoxin (LT and STa respectively). They are aided in their pathogenesis by adhesins [also called colonization factor antigens (CFAs)] consisting of specialized pili which facilitate bacteril colonization the small intestine. CFAs, including CFA/I, CFA/II (CS1, CS2, CS3) and CFA/VI (CS4, CS5, CS6) are produced by ETEC strains causing the vast majority of severe ETEC diarrheal episodes. Our vaccine targets these two toxins and the 7 most common adhesins, and is unique in several respects. It delivers these antigens in a single strain, it includes non-toxic antigens to STa, and to the active as well as the binding subunit of the LT toxin, it will hae improved immunogenicity due to the adjuvant activity of the mutant LT as well as its direct binding to the host small intestinal receptor GM1 through the LT binding subunit. We will develop this new ETEC vaccine by creating fusion proteins which incorporate the key adhesins with the ST and LT toxins. We hypothesize that a chimeric CFA antigen carrying representative epitopes from CFA/I, CS1-CS6 adhesin induces anti-adhesin immunity cross protecting against adherence from each adhesin, that a fusion of this multiepitope CFA antigen with a STa13-LT192A2:B toxoid fusion induces broad anti-adhesin immunity and antitoxin immunity against both toxins, and that this adhesin-toxoid fusion can be expressed at a native LT-like holotoxin-structure leading a strong local mucosal immune response. Our unique expertise with pig and rabbit models allows us to unambiguously validate the immunogenicity and efficacy of the new candidate vaccine. Results from this study will lead toward development of an affordable vaccine for ETEC diarrhea with potential to save thousands of children each year -- with a public health impact comparable to rotavirus vaccine.